A novel mutant-allele capture method for enrichment of low abundant cancer mutants.

Abstract

e14632 Background: High sensitivity detection of cancer mutants can facilitate cancer diagnosis, treatment selection, and disease monitoring. Improved methods to detect low abundance of cancer driver mutants are needed. Methods: We developed a novel method, termed PEAC, with a super high sensitivity to detect low abundant mutant from cancer driver genes through mutant capturing followed by Sanger sequencing. Circulating free DNA (cfDNA) standards containing 20 ng of 5%, 1%, 0% (wild-type), or 40 ng of 0.1% cancer driver mutants were used to establish the method. After pre-amplification of both wild-type and mutant alleles, mutant fragments were specifically bound by biotin-labelled, locked–nucleic acid modified probes, enriched by streptavidin beads, amplified through PCR and sequenced using Sanger for detection. Results: The wild-type standard did not show any mutation signal either with or without PEAC, nor did the standards with 5%, 1%, or 0.1% mutants prior to PEAC enrichment. However, after PEAC enrichment, 5%, 1%, and 0.1% of cancer driver mutants ( EGFR L858R, EGFR T790M, and BRAF V600E) predicting sensitivities to target therapies were enriched to ~90%, ~80% and 30-40% abundance, respectively. In addition to the application for point mutations, PEAC methodology was also used to enrich deletion mutant; 5%, 1%, and 0.1% of EGFR exon 19 deletion, E746_A750del, were all enriched to 90-100% frequency after PEAC. To address the limit of detection of PEAC technology, 100 ng of cfDNA standard containing 0.01% EGFR L858R and EGFR exon 19 deletion mutant were enriched to 15% and 50%, respectively, after PEAC, which indicates up to 5000-fold enrichment for EGFR exon 19 deletion by PEAC technology. Conclusions: PEAC technology can enrich very low abundance of key cancer driver mutants predicting sensitivities to tyrosine kinase inhibitors. PEAC enrichment in combination with Sanger sequencing may guide the application of cancer target therapy and represent a useful technology in the era of precision medicine.